This proposal entitled "Biochemical Mechanisms of Insulin Receptor Endocytosis" focuses on the regulation of insulin receptor endocytosis. The elucidation of insulin action at the cellular level is a significant component of attempts to understand Type II diabetes. Insulin receptor internalization is deficient in some insulin-resistance states and may play an important role in the physiology of insulin action; furthermore, the internalization of insulin-insulin receptor complexes in the liver is one of the major physiological mechanisms for the clearance of serum insulin. this proposal will identify structural motifs in the insulin receptor which allow it to enter the endocytic system, and will identify the intracellular proteins which mediate this recognition. The insulin receptor contains two tyrosine residues in the cytoplasmic juxtamembrane region which are present in the sequences GPDY953 and NPEY960; structural analysis both tyrosine residues are in beta-turns; mutagenesis studies suggest that these tyrosyl/beta-turns serve as recognition motifs for insulin receptor internalization. This hypothesis will be tested by constructing mutant receptors in which this tyrosine/beta-turn motif is specifically maintained or disrupted. to further define regions of the receptor involved in endocytosis, domain-specific anti-receptor antibodies and receptor-derived peptides will be microinjected into intact cells. Finally, experiments will be conducted to identify the proteins which regulate insulin receptor endocytosis, using affinity chromatography techniques based on our knowledge of endocytosis-specific receptor domains. These studies will substantially add to our understanding of the mechanisms which govern the endocytosis of the insulin receptor as well as other cell surface receptors.